Novel nuclear transfer technique through the processes of enucleation after activation to produce cloned mammals

Abstract

The present invention relates to a method for producing a mammal reconstruction cell, a method for producing a nuclear transferred (NT) embryo capable of developing into a non-human mammal, a method for producing a non-human mammal fetus, a method for producing a non-human mammal and the products prepared by the foregoing methods.

1 . A method for producing a mammal reconstruction cell comprising: 
 (a) providing a mammalian oocyte;    (b) providing a mammalian donor cell;    (c) transferring the donor cell or the nucleus thereof into the oocyte;    (d) fusing the donor cell or the nucleus thereof with the oocyte to generate a tetraploid cell;    (e) activating the tetraploid cell; and    (f) enucleating the oocyte in the activated tetraploid cell to generate a diploid reconstruction cell.    
   
   
       2 . The method as claimed in  claim 1 , wherein the mammal is livestock.  
   
   
       3 . The method as claimed in  claim 2 , wherein the mammal is bovine.  
   
   
       4 . The method as claimed in  claim 3 , wherein the oocyte provided in step (a) is matured in vivo or in vitro and contains a first polar body.  
   
   
       5 . The method as claimed in  claim 3 , wherein the donor cell provided in step (b) is a somatic cell.  
   
   
       6 . The method as claimed in  claim 5 , wherein the somatic cell is a transgenic cell.  
   
   
       7 . The method as claimed in  claim 3 , wherein the donor cell provided in step (b) is serum starved.  
   
   
       8 . The method as claimed in  claim 3 , wherein the donor cell provided in step (b) is not serum starved.  
   
   
       9 . The method as claimed in  claim 3 , wherein the step (c) is transferring the donor cell into the perivitelline space of the oocyte.  
   
   
       10 . The method as claimed in  claim 3 , wherein the step (c) is directly injecting the donor cell into the cytoplasm of the oocyte.  
   
   
       11 . The method as claimed in  claim 3 , wherein the oocyte in step (d) is unenucleated.  
   
   
       12 . The method as claimed in  claim 3 , wherein the fusion of step (d) is using an electrical stimulus.  
   
   
       13 . The method as claimed in  claim 3 , wherein the activation of step (e) is incubating the fused cell in an activating solution comprising calcium ionophore and 6-dimethylaminopurine (6-DMAP).  
   
   
       14 . A mammal reconstruction cell produced by the method as claimed in  claim 1 .  
   
   
       15 . A method for producing a nuclear transferred (NT) embryo capable of developing into a non-human mammal, the method comprising: 
 (a) providing a mammalian oocyte;    (b) providing a mammalian donor cell;    (c) transferring the donor cell or the nucleus thereof into the oocyte;    (d) fusing the donor cell or the nucleus thereof with the oocyte to generate a tetraploid NT embryo;    (e) activating the tetraploid NT embryo;    (f) enucleating the oocyte in the tetraploid NT embryo to generate a diploid NT embryo; and    (g) culturing the diploid NT embryo.    
   
   
       16 . The method as claimed in  claim 15 , wherein the mammal is livestock.  
   
   
       17 . The method as claimed in  claim 16 , wherein the mammal is bovine.  
   
   
       18 . The method as claimed in  claim 17 , wherein the oocyte provided in step (a) is matured in vivo or in vitro and contains a first polar body.  
   
   
       19 . The method as claimed in  claim 17 , wherein the donor cell provided in step (b) is a somatic cell.  
   
   
       20 . The method as claimed in  claim 19 , wherein the somatic cell is a 7 transgenic cell.  
   
   
       21 . The method as claimed in  claim 17 , wherein the donor cell is serum starved.  
   
   
       22 . The method as claimed in  claim 17 , wherein the donor cell is not serum starved.  
   
   
       23 . The method as claimed in  claim 17 , wherein the step (c) is transferring the donor cell into the perivitelline space of the oocyte.  
   
   
       24 . The method as claimed in  claim 17 , wherein the step (c) is directly injecting the donor cell into the cytoplasm of the oocyte.  
   
   
       25 . The method as claimed in  claim 17 , wherein the oocyte in step (d) is unenucleated.  
   
   
       26 . The method as claimed in  claim 17 , wherein the fusion of step (d) is using an electrical stimulus.  
   
   
       27 . The method as claimed in  claim 17 , wherein the activation of step (e) is incubating the fused tetraploid NT embryo in an activating solution comprising calcium ionophore and 6-dimethylaminopurine (6-DMAP).  
   
   
       28 . A nuclear transferred (NT) embryo capable of developing into a non-human mammal produced by the method as claimed in  claim 15 .  
   
   
       29 . A method for producing a non-human mammal fetus, the method comprising: 
 (a) providing a mammalian oocyte;    (b) providing a mammalian donor cell;    (c) transferring the donor cell or the nucleus thereof into the oocyte;    (d) fusing the donor cell or the nucleus thereof with the oocyte to generate a tetraploid NT embryo;    (e) activating the tetraploid NT embryo;    (f) enucleating the oocyte in the tetraploid NT embryo to generate a diploid NT embryo;    (g) culturing the diploid NT embryo; and    (h) transferring the cultured diploid NT embryo into a recipient mammal female so as to produce a mammal fetus.    
   
   
       30 . The method as claimed in  claim 29 , wherein the mammal is livestock.  
   
   
       31 . The method as claimed in  claim 30 , wherein the mammal is bovine.  
   
   
       32 . The method as claimed in  claim 31 , wherein the oocyte provided in step (a) is matured in vivo or in vitro and contains a first polar body.  
   
   
       33 . The method as claimed in  claim 31 , wherein the donor cell provided in step (b) is a somatic cell.  
   
   
       34 . The method as claimed in  claim 33 , wherein the somatic cell is a transgenic cell.  
   
   
       35 . The method as claimed in  claim 31 , wherein the donor cell is serum starved.  
   
   
       36 . The method as claimed in  claim 31 , wherein the donor cell is not serum starved.  
   
   
       37 . The method as claimed in  claim 31 , wherein the step (c) is transferring the donor cell into the perivitelline space of the oocyte.  
   
   
       38 . The method as claimed in  claim 31 , wherein the step (c) is directly injecting the donor cell into the cytoplasm of the oocyte.  
   
   
       39 . The method as claimed in  claim 31 , wherein the oocyte in step (d) is unenucleated.  
   
   
       40 . The method as claimed in  claim 31 , wherein the fusion of step (d) is using an electrical stimulus.  
   
   
       41 . The method as claimed in  claim 31 , wherein the activation of step (e) is incubating the fused tetraploid NT embryo in an activating solution comprising calcium ionophore and 6-dimethylaminopurine (6-DMAP).  
   
   
       42 . A non-human mammal fetus prepared by the method as claimed in  claim 29 .  
   
   
       43 . A method for producing a non-human mammal, the method comprising: 
 (a) providing a mammalian oocyte;    (b) providing a mammalian donor cell;    (c) transferring the donor cell or the nucleus thereof into the oocyte;    (d) fusing the donor cell or the nucleus thereof with the oocyte to generate a tetraploid NT embryo;    (e) activating the tetraploid NT embryo;    (f) enucleating the oocyte in the tetraploid NT embryo to generate a diploid NT embryo;    (g) culturing the diploid NT embryo; and    (h) transferring the cultured diploid NT embryo into a recipient mammal female so as to produce a mammal fetus that undergoes full fetal development and parturition to generate a live-born mammal.    
   
   
       44 . The method as claimed in  claim 43 , wherein the mammal is livestock.  
   
   
       45 . The method as claimed in  claim 44 , wherein the mammal is bovine.  
   
   
       46 . The method as claimed in  claim 45 , wherein the oocyte provided in step (a) is matured in vivo or in vitro and contains a first polar body.  
   
   
       47 . The method as claimed in  claim 45 , wherein the donor cell provided in step (b) is a somatic cell.  
   
   
       48 . The method as claimed in  claim 47 , wherein the somatic cell is a transgenic cell.  
   
   
       49 . The method as claimed in  claim 45 , wherein the donor cell is serum starved.  
   
   
       50 . The method as claimed in  claim 45 , wherein the donor cell is not serum starved.  
   
   
       51 . The method as claimed in  claim 45 , wherein the step (c) is transferring the donor cell into the perivitelline space of the oocyte.  
   
   
       52 . The method as claimed in  claim 45 , wherein the step (c) is directly injecting the donor cell into the cytoplasm of the oocyte.  
   
   
       53 . The method as claimed in  claim 45 , wherein the oocyte in step (d) is unenucleated.  
   
   
       54 . The method as claimed in  claim 45 , wherein the fusion of step (d) is using an electrical stimulus.  
   
   
       55 . The method as claimed in  claim 45 , wherein the activation of step (e) is incubating the fused tetraploid NT embryo in an activating solution comprising calcium ionophore and 6-dimethylaminopurine (6-DMAP).  
   
   
       56 . A non-human mammal prepared by the method as claimed in  claim 43.